CN1902311A - Novel recombinant proteins with n-terminal free thiol - Google Patents

Abstract

The present invention relates to novel modified proteins having N-terminal free thiols that can be produced by recombinant methods and are ready for further chemical derivatization. In particular, the invention relates to erythropoietin conjugate compounds having altered biochemical, physiochemical and pharmacokinetic properties. More particularly, one embodiment of the invention relates to erythropoietin conjugate compounds of the formula: (M)n-X-A-cys-EPO (I) where EPO is an erythropoeitin moiety selected from erythropoietin or an erythropoietin variant having at least one amino acid different from the wild-type human EPO, or any pharmaceutical acceptable derivatives thereof having biological properties of causing bone marrow cells to increase production of red blood cells; cys represents the amino acid cysteine and occurs at position -1 relative to the amino acid sequence of the erythropoietin moiety; A indicates the structure of the residual moiety used to chemically attach X to the thiol group of -1Cys; X is a water soluble polymer such as a polyalkylene glycol or other polymer; M is an organic molecule (including peptides and proteins) that increases the circulating half-life of the construct; and N is an integer from 0 to 15.

Description

New recombinant protein with N-terminal free thiol

Invention field

The present invention relates to and by recombination method production and can further carry out chemically derived new modified protein.Particularly, the present invention relates to have the erythropoietin conjugates compound of biological chemistry, physiochemistry and the pharmacokinetic property of change.

Background of invention

Erythropoietin (EPO) is a kind of glycoprotein of natural formation, and its function is to participate in the adjusting of red corpuscle synthetic as G CFS and as the main factor.Erythropoietin is by stimulating precursor cell in the marrow, impelling their divisions and differentiation to become mature erythrocyte to work.This process is strict control in vivo, like this red corpuscle in circulation destruction or removing could with the formation speeds match of new cell.Naturally occurring EPO is a kind of glycoprotein (people such as Jacobs, Nature 313 (6005), 806-810 (1985)) that produces in kidney.Like this, except the precursor in the marrow produces the low or insufficient situation of protoerythrocyte, any kidney function damage or destructive situation as the kidney disease in latter stage, have been represented the erythropoietin reaction condition.

Verified various kinds of cell type produces EPO, and except the red corpuscle progenitor cell, a lot of cell expressing EPO acceptors comprise capillary endothelial cell and brain inner cell.Stellate cell reacts on anoxic and produces EPO (Masuda; S.et al.1994J Biol Chem 269:19488-19493); and near the neurocyte external source EPO can protect is avoided ischemic injuries (the Sakanaka M. in the animal model; et al.1998; Proc Natl Acad Sci USA 95:4635-4640); therefore, EPO may and work from nerve destruction or disease recovery in neural destruction of prevention or disease.Recently; discovery erythropoietin protection retinal neurons is avoided acute ischemia reperfusion injury (Junk; et al.2002; Proc.Nat.Acad.Sci.99:10659-10664) and strengthen nerve recovery (Gorio et al. from experimental Spinal injury; 2002, Proc.Nat.Acad.Sci.99:9450-9455).The pathogenic neural status of neurone in the system that affects the nerves or spongiocyte may be because ischemic, apoptosis, necrosis, oxidation or radical damage and exitotoxicity (excitotoxicity).Europathology comprises that for example, brain and ischemia of spinal cord, acute cerebral insult, Spinal injury, retinopathy and neurodegenerative disease are as alzheimer's disease, Parkinson's disease, Huntington Chorea and ALS.Therefore, think that at present external source EPO has protection or prophylactic effect to some or all of these diseases.

In fact, EPO proved internal secretion (hormone), autocrine and paracrine function in various kinds of cell and types of organization (to the activation or the hormesis of self and adjacent cells type) (referring to, Lappin, T.R.et al., 2002, the summary of Stem Cells 20:485-492), described cell and types of organization comprise the (Parsa of cardiac muscular tissue, C.J.et al, 2003, J Clin Invest.112 (7): 999-1007) and gastrointestinal tissue (Fatouros, M.S., 2003, Eur J Surgery 165 (10): 986-992).Therefore, the potential treatment indication that gives EPO has substantially exceeded renal insufficiency and anaemia.

Utilized recombinant DNA technology to produce erythropoietin, it expresses (Lin, US 5618698) by clone's EPO gene and in Chinese hamster ovary cell.The EPO of recombinant production is as a kind of effective therapeutical agent supply for the treatment of the various forms anaemia, and described anaemia comprises and the HIV infected patient of chronic renal failure, azidothymidine in treating and the relevant anaemia of cancer patients of bone marrow depression chemotherapy.EPO glycoprotein is through administered parenterally, and it is as vein (IV) or subcutaneous (SC) injection in the conventional aqueous buffer solution of carrier containing human serum albumin (HSA).This preparation is that trade(brand)name is sold on American market with EPOGEN  and PROCRIT .These products contain the erythropoietin in single agent preservative-free of 1ml or the anticorrosion phial of 2ml multi-agent.

Although these preparations are proved to be extremely successful, some defective is also relevant with this product.At present, plasma half-life of being lacked such as the biological activity cycle of the protein therapeutic agent of erythropoietin and to the restriction of proteasome degradation susceptibility.Such as the short-half-life of the such therapeutic protein of EPO, make frequently to necessitate.This treatment to chronic condition is disadvantageous, and may cause patient dependence poor, does not therefore reach best effect.Thereby carried out the plasma half-life that very big effort increases EPO.

In recent years, the nonantigenic water-soluble polymers such as polyoxyethylene glycol (PEG) is applied to treating and diagnosing on the covalent modification of relevant polypeptide.For example, PEG is covalently bound arrives such as interleukin (Knauf, people such as M.J., J.Biol.Chem.1988,263,15,064; Tsutsumi, people such as Y., J.Controlled Release 1995,33,447), Interferon, rabbit (Kita, people such as Y., Drug Des Delivery 1990,6,157), catalase (Abuchowski, A. wait the people, J.Biol Chem.1977,252,3,582), superoxide dismutase (Beauchamp, people such as C.O., Anal Biochem.1983,131,25) and adenosine deaminase (Chen, people such as R., Biochim, Biophys.Acta.1981,660,293) on the therapeutical peptide, reported the transformation period in the body that prolongs them, and/or reduce their immunogenicity and antigenicity.

The deutero-polyethylene glycol compound just disclosed (US5438040) in the past.This translation back deutero-method is equally applicable to EPO.For example, WO94/28024 discloses a kind of polymer conjugate with the active carbohydrate modification of erythropoietin, and wherein PEG connects by oxidized carbohydrate.US 4904584 discloses the polyalkylene oxide conjugated thing of the polypeptide variants that has lacked Methionin, comprises EPO.WO 90/12874 has described the preparation of mono methoxy-PEG-EPO (mPEG-EPO), and wherein EPO has comprised a cysteine residues that is imported by genetically engineered, and specific PEG reagent is arrived above it by covalently bound.Other PEG-EPO composition is open in EP 605693, US 6,077,939, WO01/02017 and EP539167.

Applicant's common pending application USSN 09/431,861 discloses with PEG modified antibodies and antibody fragment, and has proved that PEG can increase the circulating half-life in mouse and the primate.The PEG of derivatize is used to the Fab fragment of modified antibodies c7E3.The increase of circulating half-life is directly proportional with the molecular weight of PEG.Along with the molecular weight of PEG increases, compound reduces in the ability of vitro inhibition ADP inductive platelet aggregation, and by BIAcore measure unaffected with combining of purifying GPIIb/IIIa.Add lipid acid or lipid to PEG (PEG3.4K-DSPE[DSPE]), produced the circulating half-life longer than PEG5K.Although c7E3 Fab ' (PEG5K) reduces c7E3Fab ' (PEG with respect to the external activity of c7E3 Fab _3.4K-DSPE) ₂Activity identical with c7E3Fab.

The common pending application U.S. serial 60/377 of applicant's another one, 946 disclose the method for modifying EPO, wherein EPO and nonantigenic hydrophilic polymer covalency are puted together, described polymkeric substance is connected with organic molecule, and this organic molecule makes the increase of the circulation serum half-life of composition increase manyly than adding independent hydrophilic polymer.Described method comprises making to have promoting erythrocyte and generate active albumen or glycoprotein and have the step of nonantigenic substantially functionalized hydrophilic polymer reaction that is used for polymkeric substance is connected in the linking group of glycoprotein.The preparation method comprises makes EPO and the polyalkylene oxide reaction that activates form, activate form polyalkylene oxide will with the functional group reactions on the EPO.This comprises the activated polyalkylene oxide, as active ester, hydrazides, hydrazine, Urea,amino-, thiosemicarbazide maleimide or halo ethanoyl polyalkylene oxide.

Many all often have a restricted aspect with the pure chemistry method by the method that is conjugated to PEG and goes up (" Pegylation ") modifying protein, that be exactly be present on the come-at-able lysine residue and/or proteinic N end amine on amino indifference and incomplete reaction.Other chemical process requires the oxidation of carbohydrate group as a part of modifying strategy, and this can cause not exclusively or discontinuous reaction and indefinite product composition equally.Like this, consider the present selection that has, a kind of is more favourable in method gentleness, that modify under the locus specificity mode such as the human cytokines of EPO.

Modification or interpolation motif will bring multiple risk to naturally occurring molecule, this is carrying out purpose to those is that the personnel that provide the genetic engineering technique of production method to put into practice for therapeutic protein are known.The most tangible in these effects is exactly forfeiture or part loss of biological activity.In other cases, the expression level of the expression vector of structure is for low can't the accepting of production clone.Coupling or the possible shortcoming that merges a kind of another kind of method from naturally occurring proteinic heterologous sequence are to produce epitope and cause unnecessary immune response in patient's body, and this has finally limited the long-term efficacy of therapeutic protein.In addition, utilize the chemical process attack the reactive functional group of tool Methionin to come modifying protein also to change isoelectric point of protein and pKa, this may influence proteinic structure and activity.Therefore, when purpose provides the product of safety and economic production, understand that these restrictions are very important.

The verified lysine residue that imports is the effective ways (Kuan, Chien Tsun et al.Journal of BiologicalChemistry 269,7610-7616 (1994)) that import the unique site that is used for pointed decoration on albumen.The N-terminal halfcystine has unique especially biochemical characteristic.Because α-amine is closely adjacent with the side chain thiol, N-terminal cysteine residues and ester moiety reaction form stable amido linkage (Tam, James P.et al.Biopolymers 51,311-332 (2000)).This makes peptide, albumen and other molecule put together with height selection and stable manner and proteic N-terminal.The existence of the free α-amine on the halfcystine makes local pH more alkaline, causes the N-terminal thiol higher with respect to the thiol reactivity on the inner Gelucystine.Therefore, another advantage is to carry out conjugation reaction under lower pH, causes that albumen is more nonspecific derives.The further advantage that the thiol of halfcystine is converted into thioesters is the change that it does not cause halfcystine iso-electric point or electric charge.

But in excretory albumen, cysteine residues exists as the curing Gelucystine usually, and causes the stabilization of albumen tertiary structure.Add extra cysteine residues and caused destroying proteic danger.For example, EPO contains 4 cysteine residues that participate in disulphide bridges.Therefore, there is such possibility, that is, imports the 5th cysteine residues at N-terminal and may disturb the correct folding acceptor that also therefore disturbs to discern.

When modifying excretory albumen, import amino acid at ripe N-terminal interesting challenge is provided, that is, destroy the signal sequence cleavage site.Translated a large amount of excretory albumen, it has extra zone at N-terminal, and biosynthesizing begins (being called signal or leader sequence) from here, and this district is directed at endoplasmic reticulum (ER) with albumen.Signal sequence has some feature: they have about 20-25 amino acid usually, are alkaline at N-terminal, are highly hydrophobic at the middle part, and have little not charged residue before the site that signal peptidase cuts it.Hydrophobic region is crucial for the interaction with the ER receptor complex, and the translation in the promotes oxidn environment and folding.When film is secreted, in the ripe N-terminal amino acid of proteic function enzymatic cutoff signal sequence, described amino acid becomes only free α amine in the albumen.Kept signal section, in cell, degraded.Like this, to adding amino acid with becoming the amino acid whose precursor protein sequence of new N-terminal, need insert extra residue between signal sequence and normal ripe N-terminal, therefore change has the natural cleavage site of unknown influence to cutting and secernment efficiency.People EPO precursor polypeptide has 27 amino acid whose signal sequences.In case be arranged in the ER chamber of cell, cleavable signal peptide between the L-Ala 28 of the glycine 27 of signal peptide and ripe EPO chain.

Can add amino acid or change amino acid to albumen with gene engineering method by adding or change nucleic acid coding sequence.Therefore, those skilled in the art will recognize that the employing standard technique, prepare the possibility of new treatment protein sequence by operation encoding sequence or cDNA, described treatment protein sequence has the cysteine residues that is positioned at naturally occurring N-terminal amino-acid residue N-terminal.For the proteic N-terminal that increases through engineering approaches is the possibility of halfcystine, usually, the endogenous signal sequence must can effectively be scheduled ER with protein targets by known, and produces the sequence replacement of suitable cleavage site.

For example, employing is used at yeast (US4775622 and Elliott, S.et al. (1989) Gene79,167-180) and mammalian cell (Kim, Chang H.et al. (1997) Gene199, express the substitution signal sequence of EPO 293-301), the allos signal sequence successfully is used for proteic ripe N-terminal through engineering approaches.But, also not about the allos signal sequence being used to secrete the proteic report of treatment of N-terminal engineered forms.

Summary of the invention

The invention provides a kind of composition of polypeptide conjugate (conjugate) of biologically active, wherein modified the polynucleotide sequence of coded polypeptide, put together the mating partner peptide with what generation had a N-terminal halfcystine, and be conjugated on the nonantigenic hydrophilic polymer described mating partner covalency and locus specificity, described polymkeric substance also can covalently be connected on the organic molecule, and two kinds of modifications all increase the circulation serum half-life of composition.

More specifically, one embodiment of the invention relate to as shown in the formula described EPO derivative

(M) _n-X-A-cys-EPO (I)

Wherein EPO is the erythropoietin part, be selected from erythropoietin or have at least one and the different amino acid whose erythropoietin variants of wild-type people EPO amino acid, or anyly have its pharmacy acceptable derivates that causes the biological property that medullary cell increases erythrocytic production; Cys represents halfcystine, and is present in respect to the aminoacid sequence of erythropoietin part-1; A represents to be used for the X chemistry is connected in-structure of the residue part of the thiol of 1Cys; X is a water-soluble polymers, as polyalkylene glycol or other polymkeric substance; M is the organic molecule (comprising peptide and albumen) that increases the circulating half-life of construct; And n is 0 to 15 integer.Can comprise other molecule between A and the X or between X and the M, to be provided for the suitable functionality of coupling or valency.Organic molecule M chooses wantonly.X is polyalkylene oxide preferably, as polyoxyethylene glycol, and also chooses wantonly.

The present invention also provides the treatment anaemia or has reduced the method for other relevant situations or the erythrocytic situation of expectation increase with endogenous erythropoietin or erythropoiesis.Method of the present invention comprises that also use combination treatment of the present invention is directly not relevant with the erythropoiesis defective, but situation that may be relevant with the anti-apoptotic effect of EPO, the anti-apoptotic effect of described EPO with keep or strengthen muscle, mucosal tissue, sexual gland function relevant with cognitive function.Method of the present invention further comprises ischemic, chemistry or the physical abuse of using composition prevention of the present invention, keeping or treat nervous tissue or other tissue.In this one side of the present invention, treatment comprises the conjugate described herein that needs the Mammals of described treatment significant quantity.As result of the present invention, active conjugate of erythropoietin that has abundant prolongation in vivo and the method for preparing described conjugate are provided.

The advantage of technology disclosed herein is that the end product that has obtained to determine is basically formed by the expression of the EPO variant of the transformation period increase that contains N-terminal cysteine residues and EPO.

The accompanying drawing summary

Fig. 1 has shown based on 166 amino acid whose people's forms and have in the square frame-aminoacid sequence of the precursor erythropoietin molecule of the through engineering approaches again of the signal sequence that 1cys residue and runic are represented.

Fig. 2 shows that the painted SDS PAGE by the N-terminal sequence of the cys-EPO of the cys-EPO of the definite purifying of chemical process and purifying analyzes, (N) expression placeholder.

Fig. 3 is the photo that shows the UT-7 cell proliferating determining result who compares cys-EPO and EPO.

Fig. 4 shows the 4-12%SDS-PAGE gel of the sample of representing in each swimming lane: (1) molecular weight marker; (2) EPO+0mM MEA; (3) EPO+0mM MEA+ maleimide-PEG; (4) EPO+15mM MEA; (5) EPO+15mM MEA+ maleimide-PEG; (6) EPO+20mM MEA; (7) EPO+20mM MEA+ maleimide-PEG; (8) EPO+25mM MEA; (9) EPO+25mM MEA+ maleimide-PEG; (10) EPO standard.

Fig. 5 shows the 4-12%SDS-PAGE gel of the sample of representing in each swimming lane: (1) molecular weight marker; (2) Cys-EPO+0mM MEA; (3) Cys-EPO+0mM MEA+ maleimide-PEG; (4) Cys-EPO+15mM MEA; (5) Cys-EPO+15mMMEA+ maleimide-PEG; (6) Cys-EPO+20mM MEA; (7) Cys-EPO+20mM MEA+ maleimide-PEG; (8) Cys-EPO+25mM MEA; (9) Cys-EPO+25mM MEA+ maleimide-PEG; (10) Cys-EPO standard.Attention is illustrated by white arrow corresponding to the band of the Cys-EPO that puts together with PEG.

Fig. 6 is the SELDI mass spectrum of EPO, EPO+0mM MEA+ maleimide-PEG and EPO+25mMMEA+ maleimide-PEG.Near 28,000 peak is corresponding to the EPO of unmodified.

Fig. 7 is the SELDI mass spectrum of Cys-EPO, Cys-EPO+0mM MEA+ maleimide-PEG and Cys-EPO+25mM MEA+ maleimide-PEG.Near 8,000 peak exists corresponding to adding 5, the peak of 960MW PEG corresponding to the EPO of unmodified.

Detailed Description Of The Invention

Albumen of the present invention is the N-cys variant of human cytokines, and has N terminal free thiol or " NTFI " that makes it possible to realize special and stable chemical modification. The albumen of considerably less natural existence or restructuring preparation is to process so that cysteine residues is the mode of the ripe N end of molecule. Method of the present invention adopts allos burst " pressures " at people's albumen maturation, naturally occurring or has the terminal cysteine that interpolation occurs of N of the engineering protein of therapeutic value. Adopt the allos burst to avoid the possibility of the inaccurate or wrong processing of the N terminal cysteine residue that needs, this situation can occur when endogenous targeting sequencing can not be compatible with described change. Bad processing or inaccurate processing cause a) the bad cutting efficiency of burst, thereby cause bad expression speed, or b) the inaccurate cutting of N end, keep like this or not cysteine, perhaps make its be retained in+2 ,+3 or+the n position. Under latter event, the specificity of albumen and the easiness of chemical modification will not as cysteine be positioned at maturation protein+ideal 1 time.

Just noticed for immunoglobulin (Ig) as far back as 1972 and to have comprised processing or " signal " propeptide albumen that target is used for the specific protein of cell exocrine surely, in recent years, the substructure of these sequences and relevant procedure of processing and enzyme (general introduction can be referring to Dalbey, et al. (1997) Protein Sci.6:1129-1138) have more been studied in great detail.

A kind of particularly preferred burst is human growth hormone (HGH) targeting sequencing (SEQ ID NO:2), still, in theory, exist many can be effectively and accurately produce the mammal allos targeting sequencing of the N end of needs. Other mammal Precursor Peptide that comprises signal peptide that produces ripe N terminal cysteine albumen is those relevant with the interferon-' alpha ' gene family. Developed in silico prediction algorithm, such as SigCleave, it is the matrix method (EMBOSS) of weighting, and SigPfam, it is based on hiding Markov model (HMM), so that predicted protein comprises the probability that signal peptide and most probable comprise cleavage site. Table 1 shows from the multi-signal sequence of people's precursor protein with the 3rd edition (www.cbs.dtu.dk/services/SignalP/ of SignalP; Bendtsen et al.J.Mol.Biol., 340:783-795,2004) cleavage site with as the N-Cys-EPO coupling of the maturation proteins that need the time of prediction. The 3rd edition Swiss-Prot database from the cleavage site of experimental verification of SignalP provides the neutral net (NN) of cultivating at eukaryotic protein and HMM is provided. Based on these predictions (table 1), show that the natural EPO targeting sequencing of prediction is not suitable as the targeting sequencing of N-Cys-EPO, and prediction hGH targeting sequencing and some, but not all interferon (IFN) protein signal peptide produces the albumen with N end Cys.

Table 1

Burst	Length	Protein name	The NCBI number of obtaining	SignalP 3.0 (NN) most probable	Signa lP3.0 (HM M) probability
						MGVHECPAWL   WLLLSLLSLP   LGLPVLG(SEQ ID   NO：1，1-27)	27	EPO	P01588	28 and 29:LGC-AP	0.476 between 27 and 28
MATGSRTSLL   LAFGLLCLPW   LQEGSA(SEQ ID NO：   2)	26	Growth hormone, isomers	NP_00050	6	26 and 27:GSA-CA							0.490 between 27 and 28
						MAWVWTLLFL   MAAAQSIQA   (SEQ ID NO：3)	19	Antibody HC		19 and 20:IQA-CA	0.777 between 19 and 20
MGIKMETHSQ   VFVYMLLWLS   GSVEG(SEQ ID NO：4)	25	Antibody LC		25 and 26:VEG-CA	0.878 between 25 and 26
						MASPFALLMV   LVVLSCKSSCSLG   (SEQ ID NO：5)	23	IFNα1	NP_07691   8	23 and 24:SLG-CA	0.324 between 23 and 24
MALTFALLVA   LLVLSCKSSCSVG   (SEQ ID NO：6)	23	IFNα2	NP_00059   6	SVG-CA	0.333 between 20 and 21
						MALSFSLLMA   VLVLSYKSICSLG   (SEQ ID NO：7)	23	IFNα4，10   &17	NP_06654   6	23 and 24:SLG-CA	0.222 between 23 and 24
MALPFVLLMA   LVVLNCKSICSLG   (SEQ ID NO：8)	23	IFNα5	NP_00216   0.	16 and 17:LNC-KS	0.205 between 22 and 23
						MALPFALLMA   LVVLSCKSSCSLD   (SEQ ID NO：9)	23	IFNα6	NP_06628   2	24 and 25:LDC-AP	0.375 between 21 and 22
MALSFSLLMA   VLVLSYKSICSLG   (SEQ ID NO：7)	23	IFNα10	NP_00216   2	Identical with IFN α 4

Burst	Length	Protein name	The NCBI number of obtaining	SignalP 3.0 (NN) most probable	Signa lP 3.0 (HM M) probability
						MASPFALLMA   LVVLSCKSSCSLG   (SEQ ID NO：10)	23	IFNα13	NP_00883   1	23 and 24; SLG-CA	0.324 between 23 and 24
MALPFALMMA   LVVLSCKSSCSLG   (SEQ ID NO：11)	23	IFNα14	NP_00216   3	23 and 24:SLG-CA	0.320 between 23 and 24
						MALSFSLLMA   VLVLSYKSICSLG   (SEQ ID NO：7)	23	IFNα17	NP_06709   1	Identical with IFN α 4
MAFVLSLLMA   LVLVSYGPGGSLG   (SEQ ID NO：12)	23	IFNδ1	P37290	23 and 24:SLG-CA	0.815 between 23 and 24
						MALLFPLLAA   LVMTSYSPVGSLG   (SEQ ID NO：13)	23	IFNΩ1	NP_00216   8	23 and 24:SLG-CA	0.649 between 23 and 24

Multiple mammalian expression vector has been successfully used to express recombinant protein. The composition of the example that generates by method of the present invention comprises the precursor that utilizes strong virus promoter, total Kozak sequence, the interested albumen of coding, and (the hHG signal peptide: the single expression vector of DNA EPO), six His marks, terminator codon and polyadenylation signal, described polyadenylation signal derive from for example SV40 (simian virus) polyadenylation signal or BGH polyadenylation signal.

In case made up the expression vector that contains composition of the present invention, just adopted the conventional method of transfection host cell to express new albumen. Transient transfection or stable transfection method can be adopted, and any host cell (preferred mammal cell) that to process the mammalian signal sequence can be adopted. The example of useful host cell system is VERO and HeLa cell, Chinese hamster ovary (CHO) clone, W138,293, BHK, COS-7 and mdck cell system. Reclaim and the method for purifying protein is well known to a person skilled in the art from cell culture, comprise the code area of adding for the amino acid motif of so-called purifying " mark ", described " mark " is such as six histidines or FLAG.

When by prokaryotic expression, containing N-terminal methionine(Met) or formyl radical methionine(Met) and not glycosylated polypeptide usually is preferred just not.But, one of skill in the art will appreciate that to the invention is not restricted to use foregoing carrier and the mammalian host cell that contains mammalian signal peptide, particularly when purpose does not lie in the generation glycosylated protein.Understood and used the bacterial system that is used to express with secretory protein.For example, streptococcus aureus nuclease signal coding sequence has been used to produce the construct of proinsulin and genus bacillus, for example, referring to EP0176320A1.Multiple secretion signal peptide sequence can be used for genus bacillus, and operates, and can produce N-terminal halfcystine polypeptide of the present invention.Described secretion encoding sequence comprises, but be not limited to O-lactamase I type signal peptide sequence, subtilis levansucrase signal peptide sequence and the bacillus amyloliquefaciens subtilisin signal peptide sequence of the α-Dian Fenmei signal peptide sequence of bacillus amyloliquefaciens, bacillus cereus.Above-mentioned secretion encoding sequence can suitably be connected between the sequence of transcribing and translate activation sequence and encoding function N-terminal halfcystine polypeptide of carrier.

EPO

EPO mainly produces in kidney, its effect be by be attached to receptor dimer on the precursor cell cause erythrocytic differentiation and propagation subsequently (Livnah, people .Science1999 such as O., 283,987-990).EPO is attached on the acceptor by two bonding surfaces, and the avidity of one of them bind receptor is stronger than another one.The crystalline structure of EPO has been differentiated, and (Syed waits people .Nature 395 (6701), 511-516 (1998); Cheetham, people .Human Erythropoietin such as J.C., NMR minimized average structure, on September 8th, 1998.Protein Data Bank ID 1BUY).The crystalline structure that is attached to the EPO on its acceptor also is described (sees Stroud, R.M. and Reid, S.W., Erythropoietin complexed with extracellular domains of erythropoietinreceptor. Protein Data Bank ID1CN4).

Erythropoietin gene has 5 exons (SEQ ID NO:1) of 193 amino acid whose propolypeptides of coding.27 amino acid whose leader sequences of N-terminal excision from propolypeptide produce 166 amino acid whose functional polypeptides.But the recombinant human erythropoietin of expressing in Chinese hamster ovary cell only contains 165 amino acid, has lacked arg166.Its mechanism is undetermined, and does not know whether the round-robin erythropoietin also lacks arg166 or further C-terminal brachymemma in blood plasma.The Nucleotide of erythropoietin and aminoacid sequence all are high conservative in Mammals.

Be modified into raw material preferably human erythropoietin (SEQ ID NO.1) or the des-166Arg SEQ ID NO:1 of the EPO of biologically active form of the present invention, or other variant of known biologically active, or has other derivative that causes the biological property that medullary cell increases reticulocyte and erythrocytic production.Can obtain EPO glycoprotein or with the U.S.4 that is hereby incorporated by, 703,008 from natural origin; 5,441,868; 5,547,933; The program reorganization of describing in 5,618,698 and 5,621,080 produces.In wild-type people EPO, the 24th, 38 glycosylation sites of representing three kinds of naturally occurring N-to be connected with 83 Asn residue.The site (Ser123) that the glycosylation of these three positions is connected with an O-accounts for about 40 weight % of the recombinant epo that produces in natural and the mammalian cell cultures.The variant that has prepared genetic modification, it has more, still less or different glycosylation sites.Also the erythropoietin with the non-glycosylated form of the biologic activity that needs, low glycosylation form or high-glycosylation form can be used for composition of the present invention.Therefore can produce nonglycosylated albumen by prokaryotic organism, will carry out the nucleotide sequence that codon adapts to mammalian proteins and be used to adopt prokaryotic cell prokaryocyte, in colibacillary expression system, cause producing the ability of non-glycosylated protein product.Described method is instructed in WO00/32772.Other variant with biologic activity has also been described, they have by among the 24th, 38 or 83 the Asn any one being carried out glycosylation pattern (Yamaguchi, K., the et al. of the change that the amino acid exchange produces, 1991, J.Biol.Chem.266:20434-20439).

The method for preparing high-glycosylation EPO is instructed in WO0249673 and EP640619.The carbohydrate chain that other N-can be connected adds the rHuEPO molecule.In mammalian cell, at the consensus sequence that is used to add carbohydrate (Asn-X-Ser/Thr) carbohydrate that N-connects is connected in the polypeptide main chain, wherein X is the amino acid except that Pro or Asp.This process occurs in the endocytoplasmic reticulum, and is catalytic by membrane-bound oligosaccharides transferring enzyme.The knowledge of identification consensus sequence is by genetically engineered Shi Faxian, thus by the change that in the sequence of the DNA that will clone, make to need will be new carbohydrate connection site importing polypeptide main chain in.Certainly, consensus sequence must be added in carbohydrate and add compatible position, for example, not disturb receptors bind or do not damage the position of folding, the conformation or the stability of molecule.Erythrocyte-stimulating factor analogues NSEP adds the site by the carbohydrate with the analogue of 2 glycosylated 4 chains of success to merge in the molecule and produce.The aminoacid sequence of NESP different on 5 positions with the sequence of human erythropoietin (Ala30Asn, His32Thr, Pro87Val, Trp88Asn and Pro90Thr), make other oligonucleotide be connected the 30th and 80 asparagine residue (Elliott et al, Blood 96:82a (2000)).Disclosed high-glycosylation variant has added three glycosylation sites that extra N-connects among the open WO0181405 of patent application: 30,53 and 88; 30,55 and 114; Or 30,88 and 114.

Also described the EPO variant of the Pegylation of glycosylation change among US6583272 and the US6340742, wherein the glycosylation consensus sequence of Tian Jiaing mainly is positioned at 30,57,59,67,88,89,136 and 138.

Method to albumen through engineering approaches again

Adopt any in the certain methods well known in the art, can prepare polypeptide variants of the present invention or its functional fragment.The mutagenesis that is oriented to oligonucleotide is the known effective procedure that systematicness imports sudden change that is used for, and does not rely on their phenotype, therefore is suitable for the directed evolution method of protein engineeringization.This method is flexibly, allows to import accurate sudden change, does not need to use restriction enzyme, and is relatively cheap.Reorganization and enzymatic are synthetic, comprise polymerase chain reaction and other amplification method can referring to, Sambrook et al. for example, Molecular Cloning:ALaboratory Manual, Third Ed., Cold Spring Harbor Laboratory, NewYork (2001) and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

Can implement to adopt the effectively synthetic and expression method of the mutagenesis synthetic mutant polypeptide that is oriented to oligonucleotide, and this method is well known in the art, be described in Adelman et al., (1983) DNA, 2:183 and Kunkel Proc Natl.Acad.Sci.USA, 82:488-492 (1985) is hereby incorporated by.

Equally, for example, can use the amino acid whose oligonucleotide of encoding mutant, for example (QuikChange TM for example, those that use in Stratagene) produce single or multiple amino acid mutations to the site-directed mutagenesis of PCR-based.Also can be with the Higuchi et al. that is hereby incorporated by, the technology of describing among the Nucleic, Acids Res.16:7351-7367 (1988) realizes the site-directed mutagenesis of encoding wild type or the proteic cDNA of parent.In general, this is crossed range request and uses two groups of primers: first pair of primer is positioned at the flank of the proteic global cDNA that will suddenly change, and therefore will produce the total length copy of cDNA when pcr amplification, and second pair of primer is complimentary to one another, and contains the sudden change of needs.Two groups of products of the initial generation of these primers, one group has the sudden change that imports near 3 ' end, and another group has the sudden change that imports near 5 ' end.But because these two kinds of products are complimentary to one another, and with the complementation of PCR primer, two groups of products can be formed on the eclipsed duplex that both direction extends.Like this, the pcr amplification of cDNA in the presence of two groups of primers can be used to produce the full-length cDNA (SEQ ID NO:14) of the construct of coding needs shown in Figure 1.

Synthesizing of gene or gene fragment, or be that acellular preparation method also is well known in the art at least.By order anneal oligonucleotide and and the method for synthetic large nucleic acids polymkeric substance can referring to, for example, the PCT of Evans application No.WO99/14318 and U.S. Patent No. 6,521,427.Be used for people EPO in the past with the synthetic gene that changes of in vitro method or the method for complete encoding sequence: for example, referring to US159687 and US6537746.The method of EPO that mutagenesis has the character of change with generation in the body is instructed in EP0843725 and US6444441.

Can implement of the present invention halfcystine to be added on activated protein with proteic any variation with the biologic activity that needs or mutant form, as the method for the N-terminal of EPO or EPO variant polypeptide.This method can go up at the glycoprotein, particularly EPO of expressing with eukaryotic cell system and implement, and perhaps can implement on not sialylated or not glycosylated albumen or EPO.

Water-soluble polymers

A kind of particularly preferred water-soluble polymers is a kind of in the several kinds of PEG.PEG comprises a basic carbon unit, HO-(CH ₂) ₂-OH, and sell with various forms by following title: polyoxyethylene glycol (various molecular weight); PEG200; PEG (various molecular weight); Polyethylene oxide; Carbowax; Alpha-hydro-omega-hydroxypoly (oxygen-1); The 1 of ethoxylation; Soxylat A 25-7 (Polyoxyethylene ether); Emkapol 200; Gafanole 200; Pluriol e 200; Polydiol 200; Polyox WSR-301; Macrogol and PolyoxyethleneIn.Used based on aspect the polymkeric substance of PEG of the present invention, they preferably have from about 200 to about 100,000 daltonian molecular-weight average, and preferably from about 2,000 to about 20,000 dalton.2,000 to 12,000 daltonian molecular weight are most preferred.

Other water-soluble polymeric material has comprised the material such as dextran, polyvinylpyrrolidone, polysaccharide, starch, polyvinyl alcohol, polyacrylamide or other similar non-immunogenic polymkeric substance.Those of ordinary skills can understand, and are foregoing only illustrative, do not plan to be limited in the type of the non-antigen polymkeric substance that is suitable for here.

Give the organic molecule of the interior transformation period of pharmacokinetics body of prolongation

Organic moiety can be connected on the hydrophilic polymer, comprises the transformation period of carbohydrate, isocyclic compound (as steroid), heterogeneous ring compound (as alkaloid), amino acid chain, protein, enzyme, enzyme cofactor or the VITAMIN of the monoesters or the monoamide of lipid acid, dicarboxylic acid, dicarboxylic acid, the lipid that contains saturated fatty acid, the lipid that contains unsaturated fatty acids, the lipid that contains saturated and unsaturated fatty acids acid mixture, simple carbohydrate, complexity with increase.

In one embodiment, the hydrophilic polymer group is replaced (as described herein, for example formula I) by 1 to about 6 alkyl, lipid acid, fatty acid ester, lipid or phosphatide group.Preferably, substituted hydrophilic polymer group is PEG straight chain or that contain side chain.Preferably, substituted hydrophilic polymer group is by lipid acid, fatty acid ester, lipid or phosphatide group or the terminal straight chain PEG (as the PEG diamines) that replaces of hydrocarbon.The hydrophilic polymer that is replaced by alkyl, lipid acid, fatty acid ester, lipid or phosphatide group can use suitable method to prepare.For example, modifier can prepare with the PEG diamines and activatory lipid acid (for example palmityl chloride) reaction of single protection.Products therefrom can be used for producing the modification EPO that comprises the PEG that is replaced by fatty acid group.Many other suitable synthetic schemess can use.For example, the polymkeric substance that contains amine can be coupled to lipid acid described herein or fatty acid ester, and the activatory carboxylicesters on lipid acid or fatty acid ester (as using N, N '-carbonyl dimidazoles activatory) can be coupled on the hydroxyl of polymkeric substance.Like this, just can make up many suitable straight chains or contain the poly structure of side chain, and finally connect or be modified to and contain the primary amine that can be used as trans-glutaminases amine donor with desired character.

Be applicable to that lipid acid of the present invention and fatty acid ester can be saturated or can comprise one or more unsaturated units.In a preferred embodiment, lipid acid and fatty acid ester comprise that about 6 are arrived about 40 carbon atoms, or about 8 are arrived about 40 carbon atoms.The lipid acid that is fit to be used for to modify in the method for the invention EPO comprises, n-dodecylate (C12 for example, laurate), n-tetradecanoate (C14, myristate), n-n-Hexadecane acid esters (C16, palmitate), n-octadecane acid esters (C18, stearate), n-eicosanoate (C20, Arachidate), n-docosane acid esters (C22; docosoic ester), n-melissyl ester (C30), n-tetracontane acid esters (C40), suitable-Δ 9-vaccenic acid acid esters (C18, oleic acid ester), all-cis formula Δ 5.8.11.14-eicosanoate (C20, arachidonate), suberic acid, tetradecadienoic acid, octadecadienoic acid, two dodecadienoic acids and analogue.The suitable fatty acids ester comprises the monoesters that contains straight chain or contain the dicarboxylic acid of side chain low alkyl group.Low alkyl group can comprise 1 to about 12 carbon atoms, and preferred 1 is arrived about 6 carbon atoms.Be used to modify the proteinic suitable fatty acids ester of the present invention and comprise, for example octadecanoic acid methyl esters, octadecanoic acid ethyl ester, octadecanoic acid propyl ester, dodecylic acid butyl ester, the secondary butyl ester of dodecylic acid, the dodecylic acid tert-butyl ester, dodecylic acid peopentyl ester, the own ester of tetradecanoic acid, suitable-Δ 9-octadecenoic acid methyl ester and analogue.

Preparation is used to transfer to the substrate of N-terminal Cys polypeptide

Comprise two or three or the more multicomponent composition that are connected in electrophilic reagent and can be used as the suitable substrate of puting together in the inventive method.

The preparation of substrate is preferably progressively carried out, and forms single thiol compound of reaction in final step.By the disulfide linkage and the thioester bond of reductive agent such as DTT cutting, and the thioester bond that can not cut under reductive condition can be used for composition of the present invention.

The formation of disulphide bridges is to adopt substrate or the activatory disulphide that contains thiol, promptly, PEG-(the C.Woghiren that adjacent pyridyl-disulphide is realized, B.Sharma, S.Stein, Protectedthiol-polyethylene glycol:a new activated polymer for reversible proteinmodification, Bioconjugate Chem 4 (1993) 314).Thioester bond normally adopts maleimide activatory substrate or PEG-iodo-acid amide to form.Also verified based on adding the new relatively reagent (M.Morpurgo of thiol to the two keys of PEG-vinyl sulfone(Remzaol, O.Schiavon, P.Caliceti, F.M.Veronese, Covalent modification ofmushroom tyrosinase with different amphiphic polymers forpharmaceutical and biocatalysis applications, Appl Biochem Biotechnol56 (1996) 59-72).It is known puting together organic molecule in other method of polymkeric substance, comprises the reagent that uses with the thiol reaction, for example, and acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol) etc.

Reactive group can be bonded directly to hydrophilic polymer, conjugate complex or by shank such as C1-C12 alkyl.As used herein, " alkyl " is meant hydrocarbon chain, and wherein one or more carbon atoms are optional to be replaced by the heteroatoms such as oxygen, nitrogen or sulphur.Suitable shank comprises, for example Tetraglycol 99 ,-(CH ₂) ₃-,-NH-(CH ₂) ₆-NH-,-(CH ₂) ₂-NH-and-CH ₂-O-CH ₂-CH ₂-O-CH ₂-CH ₂-O-CH-NH-.

Can be before final substrate be conjugated to proteic N-terminal halfcystine, and adopt any chemistry well known in the art or enzymatic means to connect water-soluble polymers X and lipophilic reagent M.Like this; for example; be used for coupling water-soluble polymers and organic molecule if use such as the amine reactive group that comprises electrophilic groups such as tosylate, methylsulfonyl ester, halogen (chlorine, bromine, iodine), N-hydroxy-succinamide ester (NHS), the phenylester that replaces, carboxylic acid halides, primary amine in most of the cases must be protected.Other puts together organic molecule is known to the method on the polymkeric substance, comprise use can with reagent such as maleimide, iodoacetyl, acryl, pyridyl disulfide, the 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol) and the analogue of thiol reaction.Aldehydes or ketones base functional group can be coupled on the molecule that contains amine or hydrazides, and azido-can generate phosphoramidate or phosphoric acid imine linkage with the three valent phosphors radical reaction.(such as referring to the Hermanson, G.T., Bioconjugate Techniques, Academic Press:SanDiego, CA (1996)) that this thiol reactive group is introduced that the appropriate method of molecule is known in the art.Reactive group can be bonded directly to hydrophilic polymer, conjugate complex or by connection portion such as C1-C12 alkyl.As used herein, " alkyl " is meant hydrocarbon chain, and wherein one or more carbon atoms are randomly replaced by the heteroatoms such as oxygen, nitrogen or sulphur.Suitable shank comprises, for example Tetraglycol 99 ,-(CH ₂) ₃-,-NH-(CH ₂) ₆-NH-,-(CH ₂) ₂-NH-and-CH ₂-O-CH ₂-CH ₂-O-CH ₂-CH ₂-O-CH-NH-.

The modification reagent that comprises shank can produce like this, such as by list-tertbutyloxycarbonyl-alkyl diamine (as list-Boc-quadrol, list-Boc-diamino hexane) and lipid acid are reacted when having 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form amido linkage between unhindered amina and fatty acid carboxylate ester.Can make the Boc blocking group remove the primary amine that can be coupled on another carboxylicesters from product with trifluoroacetic acid (TFA) processing to expose; perhaps can react with maleic anhydride, the resulting product of cyclisation with the activation maleimide derivatives that generates lipid acid (referring to; people's such as Thompson WO 92/16221 for example, its all instruction introduce here as a reference).

Substrate is puted together N-terminal Cys in polypeptide

In case selective polymer partly is used to be conjugated to the N-terminal cys of polypeptide and be synthetic preparation, if not activation must activate with the thiol reactive group in the position that need be connected with the N-terminal of polypeptide.

Sulfone

The route of synthesis that is used to prepare the active sulfone of polyoxyethylene glycol and related polymer is instructed in US5446090 (Shearwater), is incorporated herein by reference in full at this.According to the instruction in this patent, this method comprises at least 4 steps, and wherein sulphur combines with polymer molecule, is converted into active sulfone functional group by series reaction then.Further reaction (5) is under alkaline condition the haloalkane sulfone to be converted into vinyl sulfone(Remzaol.Show the particularly preferred reagent that is used for each step below, but also can use other reagent:

(1)

(2)

(3)

(4)

(5)

Step 1 is " activation " of available hydroxyl on the hydrophilic polymer.Usually can pass through two kinds of approach, promptly hydroxy esterification or replace in a kind of pathway activation hydroxylic moiety, those skilled in the art also can use other method.Hydroxy esterification is by with acid or acid derivative, forms reactive ester and finishes as sour halogenide and PEG reaction.Typical ester is sulphonate, carboxylicesters and phosphoric acid ester.Be applicable to that implementing sulfamic acid halide of the present invention comprises methylsulfonyl chloride and p-toluenesulfonyl chloride.

In substitution reaction, the hydroxyl of hydrophilic polymer generally is that halogenide replaces by the higher part of reactivity.For example, be expressed as SOCl on the structure ₂Thionyl chloride can with PEG reaction to form more reactive higher chloro PEG.

Step 2 is that sulphur is directly connected in carbon atom in the polymkeric substance, and forms the second sulfone or have the second sulfone derivatives of similar response characteristic.2 carbon " ethyl " parts needs, so that be provided for reaction site that thiol partly is connected with sulfone away from second carbon atom of sulfuryl group in the chain.

Step 3 relates to carbon is connected in sulfuryl group--SO ₂The limited oxidation of sulphur.There are many such oxygenants, comprise hydrogen peroxide and Sodium peroxoborate.Can use catalyzer, as wolframic acid.

In step 4, by the activation of hydroxyl or by with reactive higher group substituted hydroxy, the hydroxylic moiety that adds in the 2nd step is converted into reactive higher form, similar with the step of the 1st in the reaction sequence.

The second sulfone of the polymer activation that obtains is stable, and is separable, and is suitable for thiol selectivity linked reaction.For example, PEG chloroethene sulfone about 7 or lower pH under be stable, but at alkaline pH up under at least about the condition of pH9, being used to make thiol selectivity linked reaction favourable.The PEG vinyl sulfone(Remzaol also is stable and separable, can form the key of thiol selectivity, hydrolysis-stable.

Can in synthetic, add step 5,, be used for thiol selectivity linked reaction so that activatory second sulfone is converted into one of vinyl sulfone(Remzaol or its reactive derivative.The polymkeric substance vinyl sulfone(Remzaol than its second sulfone counterpart rapider with thiol reaction, and be lower than under about 11 the pH, in a couple of days at least, be stable to hydrolysis.

Estimate that reaction produces such product, wherein the carbon of ethyl or vinyl is left the part of final conjugate.US5446090 and instruction wherein provide any molecular weight and can be linearity or the active PEG sulfone that contains side chain, and it can be replacement or unsubstituted.The sulfone part makes it be specially adapted to difunctionality or Heterobifunctional application to the stability of hydrolysis.

Polymkeric substance vinyl sulfone(Remzaol and precursor thereof and derivative can be used to be directly connected in surface and the molecule with thiol.But more typically, Heterobifunctional group hydrophilic polymer as PEG, has the vinyl sulfone(Remzaol part in a position that is usually located at polymer ends, has different functional groups in opposite ends simultaneously.Have the sulfone part at an end, the Heterobifunctional PEG that has amine specificity part at another end can be connected for example identical or different proteic halfcystine and Methionin part.Perhaps, can mix second organic moiety described herein, to such an extent as to before the reaction of unreacted sulfone part, can form stable amine key with the Heterobifunctional chemoattractant molecule.

Can from multiple compound, select to be used for other active group of Heterobifunctional activatory PEG.For biology and biotechnology applications, usually can select substituting group from reactive moieties, described reactive moieties is generally used for the PEG chemistry with activated PEG, as aldehyde, trifluoro esilate (being also referred to as tresylate sometimes), n-hydroxysuccinimide eater, cyanuryl chloride, cyanuric fluoride, acylazide, succinate, right-the diazonium benzyl, 3-(right-the diazonium phenoxy group)-hydroxyl propoxy-, maleimide etc.

Also can peptide, albumen, PEG and other polymkeric substance be connected in the EPO that contains the N-terminal halfcystine by esterification.For peptide and albumen, can synthesize or express the two, so that it contains ester moiety (preferred thioesters) at C-terminal.Under gentle aqueous conditions, thioester compound can react with the EPO that contains the N-terminal halfcystine, end product is the EPO (Tam that puts together by amido linkage that forms between the carboxyl carbon of the α of cysteine residues amino and described thioesters and described thioester compound, J.P., Proc.Natl.Acad.Sci.92,12485-12489 (1995)).

The erythropoietin examples for compounds of derivatize is:

M-PEG-A-Cys-EPO, wherein the Cys representative is with respect to the Cys of biological activity erythropoietin aminoacid sequence _-1, M is lipid, carbohydrate, polysaccharide, lipid acid, derivative of fatty acid, Fatty Alcohol(C12-C14 and C12-C18) or albumen; A represents close electric thiol reactive group, the carrier or the reaction product of preferred maleimide.

(M-PEG) ₂-A-Cys-EPO, wherein the Cys representative is with respect to the Cys of biological activity erythropoietin aminoacid sequence _-1Wherein M-PEG esterification on A is two different carboxyls, and A further comprises the close electric thiol reactive group of representative, the carrier of preferred maleimide or the part of reaction product; And wherein M is lipid, carbohydrate, polysaccharide, lipid acid, derivative of fatty acid, Fatty Alcohol(C12-C14 and C12-C18) or albumen.Also comprise more high-grade polymer.

(M-PEG) ₂-R-A-Cys-EPO, wherein the Cys representative is with respect to the Cys of biological activity erythropoietin aminoacid sequence _-1Wherein A represents close electric thiol reactive group, the carrier or the reaction product of preferred maleimide; Wherein (M-PEG) ₂-R is connected in two different carboxyls on the A; Wherein M is lipid, carbohydrate, polysaccharide, lipid acid, derivative of fatty acid, Fatty Alcohol(C12-C14 and C12-C18) or albumen, and R is the construct of enhancing valency.Also comprise more high-grade polymer.

M-A-Cys-EPO, wherein the Cys representative is with respect to the Cys of biological activity erythropoietin aminoacid sequence _-1Wherein M is albumen or peptide, and A is the free cysteine side chain on described albumen or the peptide.

M-A-Cys-EPO, wherein the Cys representative is with respect to the Cys of biological activity erythropoietin aminoacid sequence _-1Wherein M is a lipid, and A represents close electric thiol reactive group, the carrier or the reaction product of preferred maleimide.

M-A-Cys-EPO, wherein the Cys representative is with respect to the Cys of biological activity erythropoietin aminoacid sequence _-1Wherein M be vitamin H, dansyl or other to EPO give can be used to study, the part of the biophysical properties of diagnosis or therapeutic purpose; And wherein A represents close electric thiol reactive group, the carrier or the reaction product of preferred maleimide.When vitamin H or another part with known binding partners are introduced in this conjugate, expect that described conjugate can be used for research, diagnosis or treatment with the form of mixture binding partners known with it such as vitamin H-avidin mixture.

M-A-Cys-EPO, wherein the Cys representative is with respect to the Cys of biological activity erythropoietin aminoacid sequence _-1Wherein M be vitamin H, dansyl or other to EPO give can be used to study, the part of the biophysical properties of diagnosis or therapeutic purpose; And wherein A represents ester or thioester group and Cys _-1Between reaction product.

The treatment administration

NTFT erythropoietin conjugates of the present invention is suitable as the hemopathic parenteral administration of treatment, described hemopathic be characterized as low or defective red corpuscle generation, as various forms of anaemias, comprise the anaemia that is associated with the cancer patient of the HIV infected patient of chronic renal failure, azidothymidine in treating and chemotherapy.It also can be applied to treat various disease states, the erratic state of imbalance and hematology aspect, for example reaping hook cytopathy, β-thalassemia, cystic fibrosis, gestation and menoxenia, early stage anaemia, Spinal injury, space sickness, acute bleeding, aging and the similar conditions early sent out.It also can be applicable to expectation increases erythrocytic situation, for example preoperative patient.Preferably, NTFT erythropoietin conjugates of the present invention gives (for example IV, IM, SC and IP) through parenteral.

Effective dose is expected to carry out the change of certain degree according to the situation and the route of administration of treatment, but is expected at the active substance of 0.1 to 100 μ g/kg (about 7 to 7000U) body weight.The preferred dose of treatment anaemia situation is about 50 to about 300 units/kg on every Wendesdays time.

NTFT erythropoietin conjugates preparation of the present invention can be used for the treatment of europathology, those of central nervous system particularly, comprise, but be not limited to: brain and ischemia of spinal cord, acute cerebral insult, Spinal injury, retinopathy and neurodegenerative disorders, as alzheimer's disease, Parkinson's disease, Huntington Chorea and ALS.Except that europathology, EPO preparation of the present invention can be used for treating because other tissue injury that ischemic or oxygen deprivation stress such as myocardial infarction cause, operation get involved the soft tissue injury that causes, comprise reticular tissue and organ damage and because the tissue injury that wound and immune-mediated inflammation cause, or promote the healing of these damages.

Pharmaceutical composition

Therapeutic NTFT albumen prepared in accordance with the present invention can turn to the pharmaceutical composition that is suitable for injecting by method commonly known in the art with pharmaceutically acceptable carrier or excipient preparation.For example, among WO97/09996, WO97/40850, WO98/58660 and the WO99/07401 suitable composition has been described.The preferred pharmaceutically acceptable carrier that is used to prepare product of the present invention comprises human serum albumin, human plasma protein fraction matter etc.Compound of the present invention can wherein contain preparationization in osmotic pressure regulator such as the 132mM sodium-chlor in the pH value is 10mM sodium phosphate/potassium damping fluid of 7.Randomly, pharmaceutical composition can contain sanitas.Pharmaceutical composition can contain the erythropoietin product of different amounts, as 10-2000 μ g/ml, as 50 μ g or 400 μ g.

The antioxidant of interpolation such as tocopherol, butylated hydroxyl toluene, butylated hydroxyanisol, ascorbyl palmitate or such as the edetate of disodium ethylene diamine tetraacetate, the stability of said composition can further be strengthened, and wherein edetate is extra in conjunction with the heavy metal that may exist.In addition, said composition can also be come enhanced stability by adding sanitas, for example phenylformic acid and the p-Hydroxybenzoate as methyl p-hydroxybenzoate and/or propylparaben.

To being characterized as hemopathic treatment low or that defective red corpuscle produces

On the one hand, the administration of NTFT erythropoietin conjugates of the present invention causes erythrocytic formation in the human body.Therefore, the administration of NTFT erythropoietin conjugates replenishes or has replaced the proteinic function of the very important natural EPO of erythrocytic generation.The pharmaceutical composition that contains the NTFT erythropoietin conjugates can be mixed with active strength, be enough to deliver medicine to by the whole bag of tricks and just stand to be characterized as hemopathic people patient low or that defective red corpuscle produces, it is independent or as the part of a kind of situation or disease that this low or defective red corpuscle produces.Can be by giving pharmaceutical composition such as injecting methods such as subcutaneous injection, intravenous injection or intramuscular injection.The mean vol of NTFT erythropoietin conjugates may change, especially should be based on the doctor's that license is arranged suggestion and prescription.The accurate amount of conjugate will be paid the utmost attention to such as the factor of being graded by other one-tenth of definite type, the situation of being treated patient and the composition of treatment situation.Such as, the every kg body weight of 0.01 to 10 μ g, the every kg body weight of preferred 0.1 to 10 μ g can be administered once in a week.

On the other hand; the purposes of NTFT erythropoietin conjugates preparation of the present invention is the human patients of getting involved at needs; with protection, recovery or reinforcement nervous tissue; the nervous tissue of central nervous system particularly; and because the function of minimizing, destruction or loss that following disease causes: brain and ischemia of spinal cord, acute cerebral insult, Spinal injury, retinopathy and neurodegenerative disorders, as alzheimer's disease, Parkinson's disease, Huntington Chorea and ALS.The purposes of NTFT erythropoietin conjugates preparation of the present invention also can be the human patients that treatment needs intervention; with protection, recover because other tissue injury that ischemic or oxygen deprivation stress such as myocardial infarction cause, operation get involved the soft tissue injury that causes; comprise reticular tissue and organ damage and because the tissue injury that wound or immune-mediated inflammation cause, or promote its healing.

Among the application with reference to various publications.It is in order to describe the state of prior art more fully here that the disclosure of these publications is introduced.

The present invention further sets forth by the following examples, and the existence of these embodiment is a purpose with illustration The compounds of this invention and preparation of compositions, rather than restrictive.

Embodiment 1

Clone cys-EPO

The N-terminal of EPO does not participate in receptors bind, and the EPO-receptor complex is left in its position.Therefore, the N-terminal of EPO provides the ideal position that is used to mix chemically modified, and this modification should have three-dimensional influence to receptors bind at least, and is therefore also influential to biological activity.Therefore, import cysteine residues at N-terminal and make it possible to EPO is carried out pointed decoration, and do not destroy receptors bind.

Therefore, by operation EPO gene order or cDNA, prepared the hEPO that has at the cysteine residues of the N-terminal of alanine residue.But, in silico analyzes prompting and only adds cysteine residues in EPO precursor encoding sequence, the cleavage site of inferring in the signal peptide is upstream moved between 24 proline(Pro) and 25 s' the Xie Ansuan, make val25 become new N-terminal residue, or cause cutting between cys (1) and the ala1, (SignalP 3.0 with the halfcystine of removing interpolation effectively fully; Www.cbs.dtu.dk/services/SignalP/).For the N-terminal of the EPO that increases through engineering approaches is the possibility of halfcystine, proposed with known can be effectively with the peptide of protein targets due to endoplasmic reticulum, preceding 26 the endogenous hEPO signal peptide of amino acid replacement (Morris as human growth hormone, A.E.et al. (1999) Journal of BiologicalChemistry 274,418-423).When by the computer mould type analysis, predict that (Fig. 1 SEQ.ID.NO:3) produces the maturation protein with N-terminal halfcystine to this heterologous protein.

In order to realize being used to being expressed in the production of construct that N-terminal has the halfcystine of interpolation, with the human growth hormone leader sequence with halfcystine codon through engineering approaches to being used for expressing new proteic carrier.

Nucleotide sequence from pEG15 amplification EPO.Nucleotide sequence from the carrier amplification hGH signal sequence that is derived from XGH5 (Nichols Diagnostic).The hGH-EPO construct is connected in the plasmid that is called the pSUE plasmid.The method of the polynucleotide of the polypeptide that is used for preparation coding SEQID NO:3 (Fig. 3) has hereinafter been described.

Design of primers

The 107bp fragment that (SEQ ID NOS:15 and 16) preparation is contained HindIII-Kozac-hGH-CYS-ApaLI with a PCR primer.

5’.HindIII.Kozac.hGH 5’-ATG CAA GCT TGC CAC CAT GGC TACAG-3’

3’.hGH.cys.ApaLIB 5’-GTG GTG GTG CAC AGG CAC TGC CCTC-3’

The 518bp fragment that (SEQ ID NOS:17 and 18) preparation is contained CYS-ApaLI-EPO-BamHI with the 2nd PCR primer.

5’.cys.ApaLI.EPO 5’-ATG CGC ATG TGC ACC ACC ACG CCTC-3’

3’EPO.BamHI 5’-GCA TGG ATC CTC TGT CCC CTG-3’

The clone

The hGH signal sequence fragment (SEQ IDNO:2) that preparation is used for final construct with first primer.The PCR gradient condition is 95 ℃ * 2 minutes, is 30 round-robin 95 ℃ * 2 minutes then, 50-60 ℃ 30 seconds, 72 ℃ 30 seconds, be 72 ℃ of down final extensions 3 minutes then, keep under 4 ℃ then.With second primer to preparing the EPO fragment of final construct.The PCR condition be 94 ℃ 2 minutes, be 30 round-robin 94 ℃ * 30 seconds then, 60 ℃ * 30 seconds, 72 ℃ * 3 minutes was 72 ℃ of down final extensions 7 minutes then, kept under 4 ℃ then.After the amplification, each PCR reactant of 10 μ L is mixed with 1 μ L, 10 * application of sample dyestuff, and (CA) (Bio-Rad, Hercules CA) go up electrophoresis at the 1%SeaKem gel for Invitrogen, Carlsbad with the 1Kb ladder.HGH PCR is reflected at the band that has produced under each temperature in the migration of about 100bp position; Downcut this band and combine, (Qiagen, Valencia CA) extract, and at 30 μ L dH according to the QIAQuickGel column extractor ₂Wash-out among the O.Similarly, downcut and extract the EPO band that moves in about 500bp position.

With BamHI and ApaLI digestion EPO fragment, with HindIII and ApaLI digestion hGH fragment, with HindIII and BamHI digested vector pSUE.By mentioned above, 10 μ L pSUE digests electrophoresis on the 1%SeaKem gel.Downcut the carrier strap at about 10Kb place, according to extraction mentioned above.(New England BioLabs, Beverly MA) handle whole 30 μ L eluates, then according to the specification sheets purifying of QIAQuick PCR purification column (Qiagen), and at 30 μ L dH with calf intestine alkaline phosphatase ₂Wash-out among the O.With QIAQuick PCR purification column purifying fragment digest, at 30 μ L dH ₂Wash-out among the O.

(Roche Applied Science, Indianapolis IN) connect each fragment and carrier to connect test kit fast with Roche.Ligation is transformed into TOP10 OneShot chemoreception attitude cell (Invitrogen), and bed board (Teknova, Half Moon Bay is on Luria-Bertani CA) (LB) flat board containing 100 μ g/mL penbritins.Each colony is selected in the selected liq LB substratum, and the 225rpm oscillating growth spends the night under 37 degree.Extract plasmid DNA with QiagenSpin Miniprep test kit (Qiagen), and be eluted to 75 μ L dH ₂Among the O.Digest all clones with restriction enzyme, be used for inserting with screening.With fluorescence dye-terminator with have the ABI3 100 genetic analysis instrument of inside primer of carrier (Applied Biosystems, Foster City CA) check order to positive colony.Two kinds of positive colonies have been identified by order-checking; But, these the two kinds sudden changes (Q22R) that all contain in the hHG signal sequence.The cutting at the C-terminal halfcystine place of this not impact prediction that suddenlys change.Final plasmid is called pSUEcysEPO.

In this research, we have adopted simple expression vector, and this carrier utilizes strong virus promotor, total Kozak sequence, interested gene (EPO), six His marks, terminator codon and derives from the polyadenylation signal of bovine growth hormone gene.Perhaps, can prepare the stable mammal cell line of expressing cys-EPO, with the expressing gene product.Adopt the HEK293E cell, still, also can use any host cell that to process the mammalian signal sequence (preferred mammal cell, but not necessarily).

Embodiment 2

The expression of cys-EPO

Express new epo protein with transient transfection, wherein DNA is absorbed by mammalian cell, is transported to nucleus and transcribes.Adopt this technology, realize the pulse of protein expression with immediate mode.After the transfection 5 days, collect product from conditioned medium, that is, and cys-EPO, and with the six His mark purifying that are positioned at the PROTEIN C end.

To encode DNA (pSUEcysEPO) transfection of cys-EPO in HEK 293E cell with cation lipid reagent (LF2K).Then, culturing cell in the serum free medium in 10 layers cell culture incubator (2934-SFMII), recovering condition substratum after 4 days is with TALONIMAC purifying cys-EPO.Dialysis and concentrate after, analyze purifying degree of purity of production (Fig. 2) by SDS PAGE, carry out N-terminal order-checking and UT-7 biological assay (Fig. 3).

Before the analysis, in biological assay, do not have having L-L-glutamic acid and 5%PBS and make hungry 24.5 hours of UT-7 cell among the IMDM of Epo.Washed cell is with 30,000 the cell bed boards in every hole.Add EPO (2.5-0.0024ng/mL) and cys-EPO (20-0.01952ng/mL) with double.47.2 after hour, 37 ℃ of cell count of measuring every hole down with Promega ' s MTS solution are carried out the OD reading with 1,2 and 3 hour interval.Income value is that average background is 0.327OD unit (Fig. 3) with the gauged background of SoftMaxPro.

Reclaim the albumen that fact proved by the nucleic acid successful expression of transfection to the cell of cys-EPO from supernatant liquor, and show by the human growth hormone signal sequence this protein targets due to endoplasmic reticulum, correct folding, and secretion.

The SDS PAGE (Fig. 2) of the coomassie brilliant blue staining of material shows tangible band at about 31KDa place, and it is fine with the 34KDa coupling that the Natively glycosylated product that is produced by people and mammalian cell causes.On the contrary, nonglycosylated EPO is about 18kDa.

The existence of the single amino acids of N-terminal order-checking proof normal mature alanine residue upstream.Show the N-terminal sequence of the material of removing from this band, being explained as follows *: because cysteine residues is not easy to be discerned by the N-terminal sequence measurement, unless this albumen is with some mode derivatize, otherwise N-terminal amino acid (N is in bracket) can only be designed to exist, promptly, by sequenator " calling ", and therefore show that albumen does not have the N-terminal of sealing.In addition, can determine that this (N) is not the C-terminal derived from the hGH signal sequence, because this is can be by the L-Ala (A) of sequence measurement evaluation.In addition, the amino acid series (N) after the calling for the first time is equivalent to sophisticated people EPO gene (APP etc.).

The nucleotide sequence of this construct of encoding is shown in SEQ ID NO:19.

Embodiment 3

The chemically modified of Cys-EPO

Experiment 1

At pH7.0 (PBS),, Cys-EPO is carried out buffer-exchanged with phosphoric acid buffer with 1mM ethylenediamine tetraacetic acid (EDTA) (EDTA).Under 37 ℃ with Cys-EPO (0.7mg/ml, be dissolved in PBS+100mM phosphoric acid salt, pH 6.8) and EPO (0.7mg/ml, be dissolved in PBS+100mM phosphoric acid salt, pH6.8) with 0mM, 15mM, 20mM and 25mM b-mercaptoethylamine (MEA) (Pierce Biotechnology, Inc., Rockford, IL) incubation 2 hours together.(Biorad Laboratories, Hercules CA) remove MEA from sample to use phosphoric acid buffer (50mM, pH 6.8) equilibrated Biospin-6 desalting column according to the explanation of manufacturers then.Then at room temperature with sample and 0.75mM maleimide-PEG (molecular-weight average: 5960) (Nektar, Huntsville, AL) incubation 1 hour together.After 1 hour, halfcystine is added to the concentration of 0.75mM, and incubation 20 minutes at room temperature.Then application of sample and on the 4-12%SDS-PAGE gel electrophoresis.Also by the SELDI mass spectrum (Ciphergen, Fulton, CA), analytic sample in the matrix that adopts sinnapic acid and on according to the anti-phase chip of H-4 of the recommendation preparation of manufacturers.Handle wild-type EPO in an identical manner.

The gel of EPO sample (Fig. 4) shows, under arbitrary condition of research, the Pegylation of obvious EPO does not take place.This shows by lacking the band of representative with respect to the molecular weight skew of the EPO standard of unmodified.The SELDI-MS of sample (Fig. 6) also shows does not have molecular weight difference, shows once more Pegylation does not take place under these conditions.

The gel of cys-EPO sample (Fig. 5) is presented at the Pegylation that tangible cys-EPO has all taken place under each condition of research.This is (to note the existence for better demonstration impurity by representative with respect to what the band (representing with white arrow) of the molecular weight skew of the EPO standard of unmodified showed, the cys-EPO standard is with higher concentration application of sample, and the molecular weight of describing skew be with respect to the observed main band of standard substance).

In the SELDI of sample mass spectroscopy (Fig. 6 and 7), near 28,000 peaks are equivalent to the EPO or the cys-EPO of unmodified.Note being equivalent to add 5 among Fig. 7, the existence at the peak of 960MW PEG, it is the proportional molecular weight difference of molecular weight with the Pegylation product of expection.Because the heterogeneity of PEG and the glycosylation on the EPO, this peak is very wide, and molecular weight must to be considered as be about.But relative molecular weight shows being connected of PEG and reduction and unreduced Cys-EPO, shows the Pegylation that cys-EPO takes place under these conditions really once more.Equally, as if the degree of Pegylation increases to some extent with respect to being used for reductive MEA concentration.This shows that at least some of the N-terminal halfcystine on the cys-EPO are and another thiol to pass through disulfide bridge connects as halfcystine or gsh, and can adopt these disulphide of condition selective reduction described herein.In sum, these data show that the N-terminal thiol on the cys-EPO is come-at-able, and can use the thiol specific reagent, modifies as maleimide-PEG.

Experiment 2

At pH7.0 (PBS),, Cys-EPO is carried out buffer-exchanged with phosphoric acid buffer with 1mM ethylenediamine tetraacetic acid (EDTA) (EDTA).Add maleimide activatory distearyl (steroyl) phosphatidylethanolamine of 3 times of molar excess in this solution, the molecular weight that wherein contains (mal-PEG-DSPE) between maleimide and the lipid is 14-20,000 polymeric joint (as PEG).In 20-25 degree centigrade of incubation reaction mixture 1 hour.Then with the reaction mixture application of sample to zorbax GF-250XL size exclusion HPLC post, with PBS with 2ml/ minute flow velocity wash-out.Merge level part of containing the modified protein peak that obtains then, the detection of biological activity.

Experiment 3

At the HOBt/HBTU of the 28.7ml 0.5M that is dissolved in dimethyl formamide (DMF) (I-hydroxybenzotriazole/2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-uronium phosphofluoric acid ester) adds 14.35ml 2M diisopropylethylamine (DIEA) in, add 5g (14.35mmol) 3-(S then, trityl)-and thiohydracrylic acid (Bachem, King of Prussia, PA).This solution is added 5gMBHA resin (0.8mmol/g) (Bachem), stirred 30 minutes.With DMF, DCM and the methyl alcohol continuous washing resin of several volumes, dry in a vacuum.The resin that obtains is used to adopt standard Boc chemistry to carry out peptide and synthesizes.With standard Boc chemosynthesis length is the peptide of 2-50 residue, finishes cutting under 90%HF, 10% p-Cresol ,-5 ℃, the condition of 1.5h.Peptide is deposited in the ether, freeze-drying, and by preparation reversed-phase HPLC (RP-HPLC) purifying.Then with the Cys-EPO+1mM EDTA incubation of peptide in being dissolved in PBS (pH 7.0) 12-48 hour.With analyzing size exclusion chromatography HPLC (SEC-HPLC) monitoring reaction, carry out sds gel electrophoresis and/or SELDI mass spectrum.Last conjugate application of sample is to Zorbax GF-250XL size exclusion HPLC post, with the flow velocity wash-out of PBS with 2ml/ minute.The level part that merges the protein peak that contains the modification that obtains then, and detection of biological activity.

Embodiment 4

The UT7 cell proliferating determining

The UT7 cell be a kind of be adapted to become the human leukemia cell line that EPO relies on (Komatsu, N. wait the people. blood (Blood) 82 (2), 456-464,1993).The rinsing 3 times in PBS of UT7 cell, and before analysis, carried out EPO hungry 24 hours.The UT7 cell is hungry in the IMDM substratum of L-glutaminate that contains interpolation and 5%FBS (I5Q).Cell in 50ml DPBS rinsing once, counting when in DPBS, suspending, and be 6 * 10 with ultimate density ⁵Individual cell/mL is suspended in (ultimate density of generation is 30000 cells in every hole) in the appropriate culture medium.The EPO standard is to be diluted to 0.85 μ g/mL (2 μ L add in the 4mL substratum) by the stock solution (1.7mg/mL) with EPO to prepare.Stock solution dilution 2: 340 is to 5ng/mL, and and then serial dilution in 1: 2 makes its concentration drop to 0.0098ng/mL in the I5Q substratum.It is the standard of 2.5ng/mL to 0.0024ng/mL that the gained diluent provides concentration.Specimen is diluted with similar method.The UT-7 cell suspending liquid sample aliquot of 50 μ L is transferred in the corresponding hole, and with flat board 37 ℃ of incubations 48 hours.Cell proliferation is to use Promega ' s MTS solution, and (Promega, Madison WI) assess, and every hole adds 20 μ L.After adding MTS1 hour, begin reading.

Fig. 3 is presented at EPO concentration of material dependency diagram in the UT7 raji cell assay Raji that carries out on the EPO of unmodified and N-terminal cys modification.The EC that calculates by the EPO of unmodified ₅₀Be 1.795 * 10 ^-11M, the EPO of modification is 2.948 * 10 ^-11M.These data show that the excretory material is activated.

Embodiment 5

In the hematopoiesis of mouse moderate stimulation

In filtering the top plastics cage under the raising condition to from Charles RiversLaboratories (Raleigh, NC) the BDF1 female mice of the about 18-20 gram of the body weight of Huo Deing divide into groups (10 in every cage).

Research the 5th day, animal is divided into 1-3 treatment group (PBS contrast, EPO and M-PEG-A-Cys-EPO), every group of 15 animals.Use CO ₂Anesthetized animal wraps blood specimen collection in the test tube of quilt at EDTA by retro-orbital sinus, and target blood volume is the 0.05mL/ sample, thereby sets up baseline values.Blood is placed in the Eppendorf tube of commercially available EDTA preparation.Aliquot sample is placed hematocrit, use the clay sealed tube, centrifugal 5 minutes.On commercially available hematocrit determination card,, obtain cell pack (PCV/ hematocrit) to the hematocrit reading.Adopt 10 μ l blood, use Coulter ^TMCounter is determined hemoglobin level.At the 0th day and the 2nd day, animals received 0.94mL (112.8mL/kg) PBS (pH 7.4), EPO (0.333/mL is dissolved in PBS) or be dissolved in M-PEG-A-Cys-EPO composition of the present invention among the PBS peritoneal injection of (its amount is equal to the biological activity of being measured calibration by the UT-7 of embodiment 4).At the 4th, 7,10,14,17 and 21 day, blood sample collection was distributed in the hematocrit with the clay sealing centrifugal 5 minutes.On commercially available hematocrit determination card,, obtain cell pack (PCV/ hematocrit) to the hematocrit reading.Adopt 10 μ l samples, use Coulter ^TMCounter is determined hemoglobin level.

Sequence table

<110>Centocor，Inc.

Cunningham，Mark

Mills，Juliane

Pool，Chadler

＜120〉has the new recombinant protein of N-terminal free thiol

<130>CEN 5046

<110>Centocor，Inc.

Cunningham，Mark

Mills，Juliane

Pool，Chadler

＜120〉has the new recombinant protein of N-terminal free thiol

<130>CEN 5046

<150>60/533617

<151>2003-12-31

<160>20

<170>PatentIn version 3.3

<210>1

<211>193

<212>PRT

＜213〉people

<220>

＜221〉signal

<222>(1)..(27)

<220>

＜221〉mature peptide

<222>(28)..(193)

<220>

＜221〉variant

<222>(193)..(193)

＜223〉brachymemma, desArg

<400>1

Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu

-25 -20 -15

Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu

-10 -5 -1 1 5

Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu

10 15 20

Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu

25 30 35

Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg

40 45 50

Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu

55 60 65

CEN5026 Cys-EPO listing.ST25.txt

Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser

70 75 80 85

Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly

90 95 100

Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu

105 110 115

Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile

120 125 130

Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu

135 140 145

Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

150 155 160 165

Arg

<210>2

<211>26

<212>PRT

＜213〉people

<400>2

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala

20 25

<210>3

<211>19

<212>PRT

＜213〉mouse

<400>3

Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln Ser

1 5 10 15

Ile Gln Ala

<210>4

<211>25

<212>PRT

＜213〉mouse

<400>4

Met Gly Ile Lys Met Glu Thr His Ser Gln Val Phe Val Tyr Met Leu

1 5 10 15

Leu Trp Leu Ser Gly Ser Val Glu Gly

20 25

<210>5

<211>23

<212>PRT

＜213〉people

<400>5

Met Ala Ser Pro Phe Ala Leu Leu Met Val Leu Val Val Leu Ser Cys

1 5 10 15

Lys Ser Ser Cys Ser Leu Gly

20

<210>6

<211>23

<212>PRT

＜213〉people

<400>6

Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys

1 5 10 15

Lys Ser Ser Cys Ser Val Gly

20

<210>7

<211>23

<212>PRT

＜213〉people

<400>7

Met Ala Leu Ser Phe Ser Leu Leu Met Ala Val Leu Val Leu Ser Tyr

1 5 10 15

Lys Ser Ile Cys Ser Leu Gly

20

<210>8

<211>23

<212>PRT

＜213〉people

<400>8

Met Ala Leu Pro Phe Val Leu Leu Met Ala Leu Val Val Leu Asn Cys

1 5 10 15

Lys Ser Ile Cys Ser Leu Gly

20

<210>9

<211>23

<212>PRT

＜213〉people

<400>9

Met Ala Leu Pro Phe Ala Leu Leu Met Ala Leu Val Val Leu Ser Cys

1 5 10 15

Lys Ser Ser Cys Ser Leu Asp

20

<210>10

<211>23

<212>PRT

＜213〉people

<400>10

Met Ala Ser Pro Phe Ala Leu Leu Met Ala Leu Val Val Leu Ser Cys

1 5 10 15

Lys Ser Ser Cys Ser Leu Gly

20

<210>11

<211>23

<212>PRT

＜213〉people

<400>11

Met Ala Leu Pro Phe Ala Leu Met Met Ala Leu Val Val Leu Ser Cys

1 5 10 15

Lys Ser Ser Cys Ser Leu Gly

20

<210>12

<211>23

<212>PRT

＜213〉people

<400>12

Met Ala Phe Val Leu Ser Leu Leu Met Ala Leu Val Leu Val Ser Tyr

1 5 10 15

Gly Pro Gly Gly Ser Leu Gly

20

<210>13

<211>23

<212>PRT

＜213〉people

<400>13

Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu Val Met Thr Ser Tyr

1 5 10 15

Ser Pro Val Gly Ser Leu Gly

20

<210>14

<211>193

<212>PRT

＜213〉people

<220>

＜221〉variant

<222>(22)..(22)

<223>Q22R

<400>14

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Arg Glu Gly Ser Ala Cys Ala Pro Pro Arg Leu

20 25 30

Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu

35 40 45

Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu

50 55 60

Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg

65 70 75 80

Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu

85 90 95

Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser

100 105 110

Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly

115 120 125

Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu

130 135 140

Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile

145 150 155 160

Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu

165 170 175

Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

180 185 190

Arg

<210>15

<211>26

<212>DNA

＜213〉people

<400>15

atgcaagctt gccaccatgg ctacag 26

<210>16

<211>25

<212>DNA

＜213〉people

<400>16

gtggtggtgc acaggcactg ccctc 25

<210>17

<211>25

<212>DNA

＜213〉people

<400>17

atgcgcatgt gcaccaccac gcctc 25

<210>18

<211>21

<212>DNA

＜213〉people

<400>18

gcatggatcc tctgtcccct g 21

<210>19

<211>606

<212>DNA

＜213〉people

<220>

＜221〉signal peptide

<222>(1)..(78)

<220>

<221>CDS

<222>(1)..(603)

<220>

＜221〉variation

<222>(64)..(66)

<223>Q22R

<220>

＜221〉N_ district

<222>(79)..(81)

＜223〉be used for the TGT codon of the Cys that N-terminal adds

<220>

＜221〉mature peptide

<222>(79)..(579)

<220>

＜221〉misc_ combination

<222>(580)..(603)

＜223〉only be used for the poly-His mark of purifying

<400>19

atg gct aca ggc tcc cgg acg tcc ctg ctc ctg gct ttt ggc ctg ctc 48

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

-25 -20 -15

tgc ctg ccc tgg ctt cga gag ggc agt gcc tgt gca cca cca cgc ctc 96

Cys Leu Pro Trp Leu Arg Glu Gly Ser Ala Cys Ala Pro Pro Arg Leu

-10 -5 -1 1 5

atc tgt gac agc cga gtc ctg gag agg tac ctc ttg gag gcc aag gag 144

Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu

10 15 20

gcc gag aat atc acg acg ggc tgt gct gaa cac tgc agc ttg aat gag 192

Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu

25 30 35

aat atc act gtc cca gac acc aaa gtt aat ttc tac gcg tgg aag agg 240

Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg

40 45 50

atg gag gtc ggc cag cag gcc gta gaa gtc tgg cag ggc ctg gcc ctg 288

Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu

55 60 65 70

ctg tcg gaa gct gtc ctg cgg ggc cag gcc ctg ttg gtc aac tcg agc 336

Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser

75 80 85

cag ccg tgg gag ccc ctg caa ctg cat gtg gat aaa gcc gtc agt ggc 384

Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly

90 95 100

ctt cgc agc ctc acc act ctg ctt cgg gct ctg gga gct cag aag gaa 432

Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu

105 110 115

gcc atc tcc cct cca gat gcg gcc tca gct gct cca ctc cga aca atc 480

Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile

120 125 130

act gct gac act ttc cgc aaa ctc ttc cga gtc tac tcc aat ttc ctc 528

Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu

135 140 145 150

cgg gga aag ctg aag ctg tac aca ggg gag gca tgc agg aca ggg gac 576

Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

155 160 165

aga gga tcc cat cat cac cat cac cat tga 606

Arg Gly Ser His His His His His His

170 175

<210>20

<211>201

<212>PRT

＜213〉people

<400>20

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

-25 -20 -15

Cys Leu Pro Trp Leu Arg Glu Gly Ser Ala Cys Ala Pro Pro Arg Leu

-10 -5 -1 1 5

Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu

10 15 20

Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu

25 30 35

Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg

40 45 50

Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu

55 60 65 70

Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser

75 80 85

Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly

90 95 100

Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu

105 110 115

Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile

120 125 130

Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Ash Phe Leu

135 140 145 150

Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

155 160 165

Arg Gly Ser His His His His His His

170 175

Claims (38)

1. the method for preparing the human cytokines conjugate, this conjugate have the polymkeric substance of the N-terminal halfcystine that is conjugated to human cytokines, and the thiol of wherein said cysteine residues participates in the formation of the covalent linkage of described conjugate, and this method comprises:

A) nucleotide sequence of the described human cytokines of acquisition,

B) select to be used for expressing described proteic signal sequence, and obtain the nucleotide sequence of described signal sequence at cell,

C) the signal sequence through engineering approaches by will (b) is to the protein sequence of (a), and codon TGT inserted makes signal sequence in the TGT upstream between the two, directed formation construct,

D) described construct is expressed in cell,

E) reclaim by described construct encoded polypeptides and

F) at the N-terminal halfcystine with described conjugation of polypeptides in polymkeric substance.

2. the process of claim 1 wherein that described orientation step is the mutagenesis that is oriented to oligonucleotide.

3. the process of claim 1 wherein that described selection step comprises the obtainable computer approach of the use public.

4. the method for claim 3, wherein possible signal sequence is selected from human growth hormone leader sequence (SEQ ID NO:2), heavy chain of antibody leader sequence (SEQ ID NO:3), light chain of antibody leader sequence (SEQ ID NO:4), human interferon δ 1 leader sequence (SEQ ID NO:12) and human interferon omega 1 sequence (SEQ ID NO:13).

5. erythropoietin conjugates has and causes medullary cell to the biological property that erythrocytic production increases, and comprises formula (M) _nPart shown in the-X-A-cys-EPO (I), wherein EPO is the erythropoietin part, be selected from erythropoietin or have at least one amino acid whose erythropoietin variants different with wild-type people EPO, or anyly has its pharmacy acceptable derivates that causes the biological property that medullary cell increases erythrocytic production, cys represents halfcystine, and is present in respect to the aminoacid sequence of erythropoietin part-1; A is the residue of thiol reactive moieties; X is a hydrophilic polymer, and chooses wantonly; M is the organic molecule that can increase the circulating half-life of described part; And n is 0 to 15 integer.

6. the erythropoietin conjugates that causes medullary cell that erythrocytic production is increased of claim 3 describedly is increased in the time ratio that gives to keep after the described erythropoietin conjugates to give time of keeping behind the unconjugated erythropoietin long.

7. the erythropoietin conjugates of claim 4, the effect of wherein keeping are because the serum half-life of the increase of the Mammals erythropoietin of unmodified relatively.

8. the erythropoietin conjugates of claim 3, wherein M partly comprises about 6 organic moiety of 1-, and it selects fatty acid group, fatty acid ester group, lipid or phosphatide independently of one another.

9. the erythropoietin conjugates of claim 4, wherein said hydrophilic polymer is a polyalkylene oxide.

10. the erythropoietin conjugates of claim 3, wherein said erythropoietin or erythropoietin partly are selected from reorganization and non-recombinant mammalian erythropoietin.

11. the erythropoietin conjugates of claim 7, wherein said polyalkylene oxide are the polyethylene oxides that replaces.

12. the erythropoietin conjugates of claim 7, wherein said polyalkylene oxide are selected from the multipolymer or the segmented copolymer of polyoxyethylene glycol homopolymer, polypropylene glycol homopolymer, alkyl-polyethylene oxide, two polyethylene oxides and polyalkylene oxide.

13. the erythropoietin conjugates of claim 7, wherein said polyalkylene oxide are molecular weight is the polyoxyethylene glycol homopolymer of about 200-about 100,000.

14. the erythropoietin conjugates of claim 3, wherein said hydrophilic polymer is linear or contains polyalkylene glycol chain, carbohydrate chain, amino acid chain or the polyvinylpyrrolidone chain of side chain, and the molecular weight of wherein said hydrophilic polymer is about 120,000 dalton of about 800-.

15. the erythropoietin conjugates of claim 12, wherein said hydrophilic polymer are linear or contain the polyalkylene glycol chain of side chain that its molecular weight is greater than 2,000 dalton.

16. the erythropoietin conjugates of claim 12, wherein said hydrophilic polymer are linearity or polyglycol chain that contains side chain or linearity or the polyglycol chain that contains the replacement of side chain, and organic moiety M is selected from alkyl, C ₆-C ₄₀Fatty acid group, C ₆-C ₄₀Fatty acid ester group, lipid groups and phosphatide group.

17. the erythropoietin conjugates of claim 14, wherein said hydrophilic polymer are linear or contain the polyglycol chain of side chain that it is by being selected from alkyl, C ₆-C ₄₀Fatty acid group, C ₆-C ₄₀The organic moiety of fatty acid ester group, lipid groups or phosphatide group is terminal to be replaced.

18. the erythropoietin conjugates of claim 15, wherein said organic moiety is a palmitoyl.

19. the erythropoietin conjugates of claim 15, wherein said organic moiety are DSPE (DSPE).

20. the conjugate of claim 3, wherein A is an ethyl, and X is PEG or other polymkeric substance, and chooses wantonly, and M be vitamin H, dansyl or other to EPO give can be used to study, the part of the biophysical properties of diagnosis or therapeutic purpose.

21. the conjugate of claim 3, wherein A is an ethyl.

22. the erythropoietin conjugates of claim 3, wherein EPO is the erythropoietin part, be selected from a) SEQ ID NO:1 from the 28th at least 165, b) have at least one amino acid whose erythropoietin variants different, or a c with SEQ ID NO:1) (a) or any pharmacy acceptable derivates (b); And cys represents halfcystine, and is present in the N-terminal position of the 28th amino acids of relative SEQ ID NO:1 or variant; A represents the residue of thiol reactive moieties; X is a hydrophilic polymer; M is alkyl, C ₆-C ₄₀Fatty acid group, C ₆-C ₄₀Fatty acid ester group, lipid groups or phosphatide group; And n is 0 to 15 integer.

23. the method for erythropoietin conjugates of preparation claim 3 comprises making the cys-EPO that has a cysteine residues at N-terminal partly contact the formula Y-X-(M) that makes up in advance _nHydrophilic polymer-organic moiety mixture, wherein Y is the thiol reactive moieties, described thiol reactive moieties contains under the condition that forms EPO-cys-polymkeric substance-conjugate or becomes residue A.

24. the method for claim 21, wherein said polymkeric substance is a polyalkylene oxide.

25. the method for claim 22, wherein said polyalkylene oxide are the polyalkylene oxides of alpha-substitution.

26. the method for claim 23, wherein said polyalkylene oxide is a polyoxyethylene glycol.

27. the method for claim 21, wherein said thiol reactive moieties is a sulfone.

28. the method for claim 25, wherein said thiol reactive moieties is the second sulfone.

29. the method for claim 21, wherein said thiol reactive moieties is a disulphide.

30. the method for claim 21, wherein said thiol reactive moieties is a maleimide.

31. the method for claim 21, wherein X is peptide or albumen, and A is Cys _-1Reaction product with thioesters or ester moiety.

32. the method for claim 21, wherein said thiol reactive moieties is an iodo-acid amide.

33. the method for claim 21, wherein A is an ethyl, and X is PEG or other water-soluble polymers, and chooses wantonly, and M be vitamin H, dansyl or other to EPO give can be used to study, the part of the biophysical properties of diagnosis or therapeutic purpose.

34. treat the method for anaemia, comprise the conjugate of the claim 3 for the treatment of significant quantity.

35. the method for claim 32, wherein said conjugate is characterised in that the serum half-life of comparing increase with the erythropoietin of puting together.

36. erythropoietin albumen or protein conjugate, contain reorganization or nonrecombinant Mammals erythropoietin, wherein added cysteine residues with free α amine by reorganization, enzymatic or chemical process, so that reactive free thiol to be provided, and described reactive free thiol does not disturb protein folding, secretion or biological activity, and described thiol can derivatize, thereby increases the proteic circulating half-life of described erythropoietin or improve the proteic biological activity of described erythropoietin.

37. the part of formula Z-cys-EPO institute formula, wherein EPO is the erythropoietin part, be selected from erythropoietin or have at least one and the different amino acid whose erythropoietin variants of wild-type people EPO amino acid, or anyly has its pharmacy acceptable derivates that causes the biological property that medullary cell increases erythrocytic production, cys represents halfcystine, and is present in respect to the aminoacid sequence of erythropoietin part-1; And Z is the allos signal sequence.

38. the part of claim 35, wherein the allos signal sequence is human growth hormone leader sequence (SEQ ID NO.2).

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